Experiments are being conducted to assess the different neuropharmacological and behavioral mechanisms underlying behavior controlled by drugs as discriminative stimuli in rats and monkeys and the ability of pharmacological or behavioral manipulations to modify such behavior. Currently, studies are focusing on delta-9-tetrahydrocannabinol (THC), the psychoactive ingredient in marijuana, the endogenous cannabinoid anandamide, methamphetamine, cocaine, nicotine and heroin. Although caffeine is a non-selective adenosine antagonist in vitro, there is no consensus about the contribution of adenosine A1 and A2A receptors to its well-known psychostimulant effects. In previous studies we found experimental evidence for a main role of A1 and A2A receptors in the motor activating effects of caffeine after its acute and chronic administration, respectively (Karcz-Kubicha et al., 2003). The predominant A1 antagonistic profile of caffeine was further demonstrated in experiments analyzing the discriminative-stimulus effects of motor-activating doses of caffeine (Solinas et al., 2005) and in experiments comparing the detailed qualitative pattern of motor activation induced by caffeine and selective A1 and A2A receptor antagonists (Antoniou et al., in press). These behavioral effects were paralleled by biochemical findings from in vivo microdialysis experiments. In those experiments either systemically or intrastriatally administered caffeine produced an increase in the extracellular concentrations of dopamine and glutamate. In agreement with the behavioral experiments, the biochemical effects were obtained after acute, but not chronic, systemic administration of caffeine (Quarta et al., 2004a, 2004b). The in vivo microdialysis experiments also suggested the existence of both A1 and A2A receptors localized in striatal glutamatergic terminals, which would be responsible for a fine-tuning modulatory role of adenosine on glutamatergic neurotransmission (Quarta et al., 2004b). We therefore decided to analyze the possible existence of A1-A2A heteromeric complexes. Immunogold detection and co-immunoprecipitation experiments indicated that A1 and A2A are co-localized in the same striatal glutamatergic nerve terminals (Ciruela et al., submitted). Furthermore, it was also shown that A1-A2A heteromers constitute a very unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A1R-A2AR heteromer that could underlie the strong tolerance to caffeine's psychomotor effects (Ciruela et al., submitted). In addition to the presynaptic A1-A2A receptor heteromer, we have previously demonstrated the existence of another heterodimeric receptor which is also a target for caffeine: the postsynaptic A2A-D2 heteromer, which is localized in the GABAergic enkephalinergic neurons (Canals et al., 2003; Ciruela et al., 2004; Ferre et al., 2004). Recently, we have shown that electrostatic interactions between a basic epitope containing adjacent Arg residues and an acidic epitope containing a phosphorylated Ser residue are involved in heteromerization of adenosine A2A and dopamine D2 receptors and heteromerization of dopamine D1 and the NR1-1 subunit of the NMDA receptor (Woods et al., 2005; Woods and Ferre, 2005). Using small peptides containing the epitopes involved in A2A-D2 receptor heteromerization we found that the Arg-phosphate electrostatic interaction posses a "covalent-like" stability (Woods and Ferre, 2005). Thus, these bonds can withstand fragmentation by mass spectrometric collision-induced dissociation at energies similar to those that fragment covalent bonds and they demonstrate an extremely low dissociation constant by plasmon resonance. We also demonstrated the importance of phosphorylation-dephosphorylation events in the modulation of this electrostatic attraction (Woods and Ferre, 2005). Thus, casein kinase I-mediated phosphorylation of the acidic epitopes from the A2A and D1 receptors makes them available to interact with the basic epitopes from the D2 receptor and the NR1-1 subunit of the NMDA receptor, respectively. On the other hand, protein kinase A-mediated phosphorylation of Ser or Thr residues adjacent to the basic epitopes of the D2 receptor and the NR1-1 subunit of the NMDA receptor slows down their attraction for the respective acidic epitopes of the A2A and D1 receptors. Most likely, the Arg-phosphate electrostatic interaction represents a general mechanism, an on/off switch for many protein-protein interactions. In another series of experiments designed to assess the contribution of endogenous opioid systems in the abuse related behavioral effects of THC we have conducted parallel in vivo microdialysis and drug discrimination investigations. Using in-vivo microdialysis techniques in freely moving rats, we found that THC produces large increases in extracellular levels of beta-endorphin in the ventral tegmental area (VTA) and lesser increases in the shell of the nucleus accumbens. We then used a two-lever choice THC-discrimination procedure to investigate whether THC-induced changes in endogenous levels of beta-endorphin regulate the discriminative effects of THC. When the opioid agonist morphine was substituted for THC, it did not produce THC-like discriminative effects, but it potentiated the discriminative effects of THC, shifting the THC dose-response curve to the left. The opioid antagonist naloxone reduced the discriminated effects of THC, confirming a facilitatory role for endogenous opioid systems in the discriminative effects of THC. Bilateral microinjections of beta-endorphin directly into the VTA, but not into the shell of the nucleus accumbens, markedly potentiated the discriminative effects of ineffective threshold doses of THC, but had no effect when given alone. This potentiation was blocked by naloxone. Altogether these results indicate that psychotropic effects of THC related to drug abuse liability are regulated by THC-induced elevations in extracellular beta-endorphin levels in brain areas involved in reward and reinforcement processes (Solinas et al. 2004). These findings suggest a novel mechanism that may underlie many previous observations of opioid system modulation of cannabinoid effects. We have also investigated which opioid receptor subtypes are involved in the discriminative effects of THC. Rats trained to discriminate THC from vehicle using a two-lever operant drug-discrimination procedure were tested with compounds that bind preferentially or selectively to either mu-, delta- or kappa-opioid receptors. The preferential mu-opioid receptor agonist heroin, the selective delta-opioid receptor agonist SNC-80 and the selective kappa-opioid receptor agonist U50488 did not produce generalization to the discriminative effects of THC when given alone. However, heroin, but not SNC-80 or U50488, significantly shifted the dose-response curve for THC discrimination to the left. Also, the preferential mu-opioid receptor antagonist naltrexone, the selective delta-opioid receptor antagonist naltrindole and the kappa-opioid receptor antagonist nor-binaltorphimine (n-BNI), did not significantly reduce the discriminative effects of the training dose of THC. However, naltrexone, but not naltrindole or n-BNI, significantly shifted the dose-response curve for THC discrimination to the right. Finally, naltrexone, but not naltrindole or n-BNI, blocked the leftward shift in the dose-response curve for THC discrimination produced by heroin. These findings suggest that mu- but not delta- or kappa-opioid receptors are involved in the discriminative effects of THC. Given the role that mu-opioid receptors play in THC's rewarding effects, the present findings suggest that discriminative-stimulus effects and rewarding effects of THC involve similar neural mechanisms.